The present invention relates to a process for detecting the malignant transformation of cells and its application for diagnosing and monitoring cancers and for developing cancer prognosis.
Chorionic gonadotropic hormone (hCG) is a glycoprotein which is formed from two subunits which are termed alpha (hCGxcex1) and beta (hCGxcex2) and which are linked non-covalently (1). During pregnancy, the trophoblastic cells of the placenta produce the dimeric hCG and the free hCGxcex1 or hCGxcex2 subunits which are found in substantial quantity in the serum. The development of sensitive and. specific techniques for assaying hCG and, independently, for assaying the free hCGxcex1 subunit and the free hCGxcex2 subunit, made it possible to show that serum levels of hCG of up to 1000 pg/ml and serum levels of the free hCGxcex1 subunit of up to 3000 pg/ml were present in non-pregnant healthy subjects (2,3). It was also observed that serum levels of hCG or of the free hCGxcex1 subunit greater than these normal values are principally to be found in patients having a testicular or ovarian tumor of trophoblastic origin.
By contrast, only low serum levels of the free hCGxcex2 subunit, i.e. which are less than 100 pg/ml, can be detected in non-pregnant healthy subjects. Serum levels of free hCGxcex2 which are greater than these normal values are found in a large number of cancer patients who are carrying a tumor of gonadal or non-gonadal origin (2). In particular, 47% of patients with cancer of the bladder, 32% of patients with cancer of the pancreas and 30% of patients with cancer of the uterine cervix have a serum level of free hCGxcex2 which is greater than 100 pg/ml (2). In patients with cancer of the bladder, the presence of an elevated level (greater than 100 pg/ml) of free hCGxcex2 is reported to be correlated with unfavorable development of the disease (4,5).
While the hCGxcex1 subunit is encoded by one single gene, which is located on chromosome 6q21.1-q23, the hCGxcex2 subunit has been reported to be encoded by a family of genes which are located on chromosome 19q13.3. Following a large number of studies, it was shown that there were 7 CGxcex2 genes, termed CGxcex27 or xcex27, CGxcex26 or xcex26, CGxcex28 or xcex28, CGxcex25 or xcex25, CGxcex21 or xcex21, CGxcex22 or xcex22 and CGxcex233 or xcex23 (6). The xcex26 and xcex27 genes are allelic (7).
Only the xcex27, xcex26, xcex28, xcex25 and xcex23 genes are able to encode the hCGxcex2 subunit, which is made up of 145 amino acids. The xcex21 and xcex22 genes are characterized by the presence:
of an insertion of approximately 770 nucleotides in the 5xe2x80x2 part of the CGxcex2 genes,
of a point mutation of the 5xe2x80x2 splicing site of the first intron of the CGxcex2 genes.
It has been shown that the p1 and xcex22 genes are capable of being transcribed in some tissues and would be able to encode a protein of 132 amino acids having a different sequence from that of hCGxcex2 (7).
Sequencing all or part of the p7, xcex26, xcex25 and xcex23 genes, and the restriction maps, showed that the nucleotides corresponding to codons 2, 4 and 117 with respect to the mature CGxcex2 protein were different depending on the genes in question (8, 9, 10).
Analysis of the nucleotide sequences for equivalent positions was carried out in the case of the xcex22 gene and it was shown that the gene exhibited the same characteristics as the xcex26 gene. On the other hand, only position 117 of the xcex21 gene was deduced by restriction map analysis and was regarded as being xe2x80x9cof the Asp typexe2x80x9d.
In addition, expression of the different CGxcex2 genes in placental tissue has been analyzed in a semiquantitative manner. The results demonstrate that the xcex25 transcripts are in much higher abundance than the xcex23 and xcex28 transcripts. The xcex27, xcex21 and xcex22 transcripts are very much in the minority (7). Finally, several studies have been carried out with the aim of looking for CGxcex2 transcripts in various normal or neoplastic nontrophoblastic tissues or on various cell lines derived from cancerous tissues of trophoblastic or nontrophoblastic origin (11-17). The techniques employed in these different studies did not distinguish between the xcex27, xcex28, xcex25 and xcex23 transcripts. These studies demonstrated that xcex27, xcex28, xcex25 or xcex23 transcripts are present, in particular, in normal testes (11), neoplastic testes (12), neoplastic bladder (13), normal placenta, choriocarcinomic placental cell lines (14-16) and in various cells lines derived from neoplastic nontrophoblastic cells (15-17).
Recently, a study was carried out on normal and neoplastic tissues of vesical origin for the purpose of quantifying the xcex27, xcex28, xcex25 and xcex23 transcripts. This study was based on the profile observed after amplifying exons 1 and 2 and enzymatically cleaving in nucleotide positions corresponding to the 5xe2x80x2 transcribed, untranslated part of exon 1 (18). This study demonstrated that normal vesical tissue only expresses the xcex27 gene and that malignant transformation of vesical tissue is accompanied by acquisition of the ability to express the xcex28 and/or xcex25 and/or xcex23 genes in 45 to 95% of cases (18).
The present invention is based on demonstrating the importance of the xcex23, xcex25, xcex28 and xcex29 genes in the progression to malignancy, in particular as compared with the expression, which exists in normal tissues, of the products of the xcex26 and xcex27 genes.
The present invention relates to a process for detecting the malignant transformation of human cells, characterized in that overexpression of the products of the xcex23, xcex25, xcex28 and/or xcex29 genes, encoding the hCGxcex2 subunit, as compared with their expression in nonmalignant cells, is demonstrated in the said cells.